Tuberculosis continues to be a major worldwide health problem and is responsible for most incidences of death by an infectious agent. The worldwide incidence of tuberculosis was estimated by the World Health Organization to be 8.8 million in 1995, with a mortality estimate of 3.0 million persons, and is expected to rise to 10.2 million by the year 2000 (Dolin, et al., Bull. WHO. 72: 213-220 (1994)). The tuberculosis problem has been compounded by the development of the AIDS epidemic and the growing number of HIV-related cases of tuberculosis (Dolin, et al., Bull. WHO. 72: 213-220 (1994)).
As the incidence of tuberculosis increases, major problems also develop concerning this disease. For example, the lack of a sensitive and rapid laboratory method of diagnosing tuberculosis makes it difficult to differentiate between M. tuberculosis and M. avium-intracellulare, both of which are frequently present in HIV infected patients. Multiple methods of detection of M. tuberculosis employing polyclonal and monoclonal antibodies have been described (Cho, et al., Yonsei Med. J. 31:333-338 (1990); Cho, et al., J. Clin. Microbiol. 30: 3065-3069 (1992); Friedman, et al., Am. Rev. Respir. Dis. 140: 668-671 (1989); Kadival, et al., J. Clin. Microbiol. 23: 901-904(1986); Mason, et al., Tubercle Lung Dis. 74:195-199(1993); Papa, et al., Res. Microbiol. 143: 327-331 (1992); Sada, et al., Lancet 2 651-652 (1983); Schoningh, et al., J. Clin. Microbiol. 28: 708-713 (1990); Verstijnen, et al., J. Clin. Microbiol. 29:1372-1375 (1991) Watt, et al., J Infect Dis. 158:681-686 (1988); Wu, et al., Chin. J. Microbiol. Immunol. 22:173-180 (1989); Yanez, et al., Clin. Microbiol. 23: 822-825 (1986)), but none have acquired a widespread role in the diagnosis of tuberculosis as these antibodies cross-react with other mycobacterial strains (Cho, et al., Yonsei Med. J. 31:333-338 (1990); Friedman, et al., Am. Rev. Respir. Dis. 140: 668-671 (1989); Kadival, et al., J. Clin. Microbiol. 23: 901-904 (1986); Wu, et al., Chin. J. Microbiol. Immunol. 22:173-180 (1989); Yanez, et al., J. Clin. Microbiol. 23: 822-825 (1986)). In addition, in order to obtain significant results, a large amount of mycobacteria or mycobacterial antigen is required (Cho, et al., J. Clin. Microbiol. 30: 3065-3069 (1992); Mason, et al., Tubercle Lung Dis. 74:195-199 (1993); Papa, et al., Res. Microbiol. 143: 327-331 (1992); Schoningh, et al., J. Clin. Microbiol. 28: 708-713 (1990); Verstijnen, et al., J. Clin. Microbiol. 29:1372-1375 (1991)). Improvements in antibody-based diagnostic tests for the detection of M. tuberculosis would require specific antibody reagents with high affinity for mycobacterial antigens. Several monoclonal antibodies have been generated against surface components of M. tuberculosis (Cho, et al., Yonsei Med. J. 31:333-338 (1990); Cho, et al., J. Clin. Microbiol. 30: 3065-3069 (1992); Mauch, et al., J. Clin. Microbiol. 26:1691-1694 (1988)) but they are often cross reactive with other strains or cytoplasmic fractions (Cho, et al., Yonsei Med. J. 31:333-338 (1990); Mauch, et al., J. Clin. Microbiol. 26:1691-1694 (1988)). There is thus a need for a monoclonal antibody that selectively binds to M. tuberculosis and does not cross react with other strains of mycobacteria.
An additional problem concerns the protocol used for detecting M. tuberculosis. The protocols described thus far for detecting mycobacteria, such as direct ELISA (Mason, et al., Tubercle Lung Dis. 74:195-199 (1993); Schoningh, et al., J. Clin. Microbiol. 28: 708-713 (1990); Verstijnen, et al., J. Clin. Microbiol. 29:1372-1375 (1991)), capture ELISA (Cho, et al., Yonsei Med. J. 31:333-338 (1990); Cho, et al., J. Clin. Microbiol. 30: 3065-3069 (1992); Friedman, et al., Am. Rev. Respir. Dis. 140: 668-671 (1989); Kadival, et al., J. Clin. Microbiol. 23: 901-904 (1986); Rattan, et al., Tubercle Lung Dis. 74: 200-203 (1993); Sada, et al., Lancet 2 651-652 (1983); Watt, et al., J Infect Dis. 158:681-686 (1988); Wu, et al., Chin. J. Microbiol. Immunol. 22:173-180 (1989); Yanez, et al., J. Clin. Microbiol. 23: 822-825 (1986)) and DOT ELISA (Cho, et al., J. Clin. Microbiol. 30: 3065-3069 (1992); Papa, et al., Res. Microbiol. 143: 327-331 (1992)), are deficient in many areas. For example, none of the methods listed above allow for visualization of single captured microorganisms. Furthermore, many of these methods require the use of polyclonal immunoglobulins which have the potential disadvantages of lot to lot variation, reliance on animal sources and unwanted cross-reactivities. Accordingly, there is an outstanding need for a method of detecting M. tuberculosis which eliminates the problems existing in current methods of detection.
The present invention provides for monoclonal antibodies, the hybridoma cell lines which produce these antibodies, and the use of such monoclonal antibodies in the detection of M. tuberculosis. More specifically, the present invention provides for monoclonal antibodies that react with surface epitopes of M. tuberculosis and the use of these monoclonal antibodies for detecting and diagnosing M. tuberculosis. 
Also provided by the present invention is a modified ELISA assay for detection of microorganisms, and a modified ELISA assay employing the monoclonal antibodies of the present invention for detecting M. tuberculosis.